Introduction: Rapid, sensitive, and specific diagnostic methods are indispensable for sexually transmitted infections (STIs). In this study, a multiplex PCR-dipstick DNA chromatography assay for diagnosis of four STI pathogens, namely Neisseria gonorrhoeae (N. gonorrhoeae), Mycoplasma hominis (M. hominis), Ureaplasma (U. urealyticum and U. parvum), and Chlamydia trachomatis (C. trachomatis), was established and evaluated. Methods: Based on the hybridization of probes and interaction between streptavidin and biotin, PCR products were visualized through hybridization of specific probes and enzymatic color generation. The sensitivity and specificity of all four pathogens were evaluated. Clinical performance of the test was evaluated using 295 specimens, and comparisons among results were determined via culture or colloidal gold assay. Results: No cross-reactions were observed, confirming the high specificity of this method. The limit of detection (LOD) of the four STI pathogens was 100 copies/mu L. The sensitivity between PCR-dipstick DNA chromatography and culture or colloidal gold assay ranged from 84.6% to 100%. The specificity was between 93.5% and 96.6%, positive predictive value ranged from 53.6% to 86.7%, negative predictive value was over 98.3%, kappa value ranged from 0.676 to 0.864 (Cohen's kappa coefficient test), and the agreement rate was over 93.5%. Conclusion: In conclusion, PCR-dipstick DNA chromatography serves as a rapid, sensitive, and specific method for simultaneous diagnosis of four STI pathogens.